Ribonucleotide reductase induced by herpes simplex type 1 virus. Characterization of a distinct enzyme.

The ribonucleotide reductase induced by herpes simplex virus type 1 (HSV-1) was purified in high yield from serum-starved baby hamster kidney (BHK-21) cells infected with HSV-1 (strain H-29). The enzyme preparation was essentially free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could cause significant depletion of substrates. The HSV-1-induced enzyme was assayed in 0.2 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-Na at the pH optimum of 8.1 and the optimal dithiothreitol concentration of 10 mM. Nucleoside diphosphates were the substrates of this enzyme. The HSV-1-induced ribonucleotide reductase was inhibited by anions. EDTA also inhibited the enzyme and this inhibition was not reversed by the addition of FeCl2. Hydroxyurea acted as a noncompetitive inhibitor versus CDP reduction (Kii = 1.3 mM Kis = 2.4 mM). The enzyme was inhibited by either free Mg2+ or free ATP. However, it was neither inhibited nor activated by the ATP X Mg complex. Reduction of either CDP or ADP was only weakly inhibited by dATP, dTTP, dGTP, and dCTP. No activation of this enzyme by these compounds was observed. The V'm values for reduction of CDP, UDP, and ADP were similar, while the GDP V'm was 2-fold greater. The K'm values were 80, 12, 1.2, and 0.65 microM for UDP, ADP, GDP, and CDP, respectively. The K'm values for CDP, GDP, and ADP were the lowest values observed for any ribonucleotide reductase. Each ribonucleoside diphosphate substrate competitively inhibited the reduction of each other substrate. The K'is values obtained for inhibition by a given ribonucleoside diphosphate were similar to the K'm value obtained for that compound as a substrate. Kinetic analysis of the combined rate of product formation when both CDP and ADP were simultaneously present as substrates produced patterns that were consistent with reduction at a common catalytic site. The 2'-deoxynucleoside diphosphate products were also competitive inhibitors versus the substrates. The K'is values versus CDP reduction were 310, 140, 9, and 5 microM for dUDP, dADP, dGDP, and dCDP, respectively. Similar K'is values were obtained when ADP was the substrate. All of these data are most consistent with the hypothesis that the HSV-1-induced ribonucleotide reductase catalyzes the reduction of all substrates at a common site. The apparent lack of significant allosteric modulation of HSV-1-induced ribonucleotide reductase, its kinetic behavior, and its low K'm for CDP, GDP, and ADP clearly differentiate this enzyme from other ribonucleotide reductases.